This research will develop enzymatic methods for the synthesis of bioactive molecules. It will focus on the following programs: 1. Develop catalytic asymmetric aldol condensations based on aldolases and variants prepared by directed evolution. 2. Develop synthesis of O- and AMinked glycoproteins in vitro by enzymatic glycopeptide ligation and in vivo in E. co//through directed evolution of tRNA and aminoacyl tRNA synthase pair. 3. Develop chemo-enzymatic methods based on glycosyltransferases reaction and programmable one-pot glycosylations for the synthesis of heparin sulfates, oligosaccharides, and glycoproteins. 4. Use genetic engineering to improve the production and specificity of synthetically useful enzymes, particularly subtilisin variants, aminoacyl tRNA synthases, sulfotransferases, glycosyltransferases, and aldolases. 5. Chemo-enzymatic synthesis of important synthons and glycoconjugates. 6. Develop new methods using enzyme products for synthesis of natural products and designed molecules, including: glycosidase and glycosyltransferase inhibitors, oligosaccharides vaccines for breast cancer and AIDS, and antibiotics. 7. Develop small D-peptides to target cell-surface D-carbohydrate epitopes using mirror image phage display (e.g., using the i-sugars prepared by the evolved aldolases to synthesize the mirror image forms (theL-sugar) of the cancer epitopes Globo H and Sialyl Tn for use in identification of L-peptides. The corresponding D-peptide will be prepared via solid phase. 8. Provide other enzymatic and non-enzymatic reactions, NMR, mass spectrometry, proteomic, glycoaarray, and modeling techniques to support these efforts in synthesis and molecular design. Significant contributions in the areas of biomedicine and synthetic organic chemistry are expected. These developments may facilitate the discovery and development of new therapeutic agents.